Four sources of long-chain polyunsaturated fatty acids (LCP) differing in their chemical structure (triglycerides or phospholipids) and in their origin (tuna triglycerides, fungal triglycerides, egg phospholipids, and pig brain phospholipids) were analyzed to determine the distribution of the component fatty acids within the molecule. Lipase and phospholipase A(2) hydrolysis was performed to obtain 2-monoacylglycerols and lysophospholipids, respectively, which allowed us to determine the distribution of fatty acids between the sn-2 and sn-1,3 positions of triglycerides or between the sn-1 and sn-2 position of phospholipids. Fatty acids in the LCP sources analyzed were not randomly distributed. In tuna triglycerides, half of the total amount of 22:6n-3 was located at the sn-2 position (49.52%). In fungal triglycerides, 16:0 and 18:0 were esterified to the sn-1,3 (92.22% and 91.91%, respectively) 18:1 and 18:2 to the sn-2 position (59.77% and 62.62%, respectively), and 45% of 20:3 n-6 and only 21.64% of 20:4n-6 were found at the sn-2 position. In the lipid sources containing phospholipids, LCP were mainly esterified to the phosphatidylethanolamine fraction. In egg phospholipids, most of 20:4n-6 (5.50%, sn-2 vs. 0.91%, sn-1) and 22:6n-3 (2.89 vs. 0.28%) were located at the sn-2 position. In pig brain phospholipids, 22:6n-3 was also esterified to the sn-2 (13.20 vs. 0.27%), whereas 20:4n-6 was distributed between the two positions (12.35 vs. 5.86%). These results show a different fatty acid composition and distribution of dietary LCP sources, which may affect the absorption, distribution, and tissue uptake of LCP, and should be taken into account when supplementing infant formulas.